Manufacturing Molecules Through Metabolic Engineering Jay D. Keasling Science 330, 1355 (2010); DOI: 10.1126/science.

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As the metabolic pathways for other potentially eclipse synthetic organic chemistry. product separation and purification) are not yl group that can be subsequently functionalized desired product. CA istry is in the production of natural products. carbon substrates).edu REVIEW Manufacturing Molecules Through Metabolic Engineering T www.sciencemag. Emeryville. and relatively little starting material is necessary so availability is not an issue. thases to form the most complicated part of the (ii) metabolic route and corresponding genes encod. bolic pathways. and enzyme strucDrugs tures allow one to query for desired strain engineering and advanced metabolic engineering. bolic engineers must weigh many trade-offs in the or metabolic intermediates may be toxic to one host including carotenoids and various plant-derived development of microbial catalysts: (i) cost and avail. Whereas existing production routes use well-known. and meta. inexpensive Alkaloids are nitrogen-containing. the range of chemicals that Polyketides and nonribosomal peptides (NRPs) have found can be produced has expanded subSucrose stantially. growth. metabolic engineering will soon rival and ways. bulk chemicals.org on December 13. metabolic engineering has been suc. and many of the bioJay D. compatible with all hosts. and metabolites in a cell to nearly infinite range of chemicals identify pathway bottlenecks. and (vi) ways to maximize yields.. by transferring product-specific enzymes or entire metabolic pathways from rare or genetically Two recent studies conclude that the large group intractable organisms to those that can be readily engineered. 1980s–early 1990s (1). titers. and production of unnatural of benzyl isoquinoline alkaloids (BIAs) will specialty chemicals. Saccharomyces cerevisiae (2). 1. new analytical tools enable the Recombination of various synthase metabolic engineer to track RNA. and synthetic chemistry to proBiomolecular Engineering. Even with these many chemically or biologically (7. extensive Fine databases of gene expression. USA. and pharmaceuticals. produc.g. products Many isoprenoids have been produced microbially. Products. some of the most valuable molecules have been produced with exist. Synthetic BiolOne area where metabolic engineering has a produce artemisinic acid. An example of using metabolic engineering and synthetic chemistry together to produce 1 Starting Materials. many more of he term “metabolic engineering” was coined in the late these valuable molecules could be produced microbially. 2011 . opening up the possibility that they may of biology aid in the design of enzymes and meta. modules allows one to produce a protein. the most complex part ogy Engineering Research Center. nutraceuticals.design decisions cannot be made independently of Isoprenoids have found use as fragrances and velopments. cerevisiae engineered to 3 National Laboratory. in part due to notable adbroad use as APIs.of the molecule. veterinary agents. Natuvances in fields adjacent to metabolic 6-C sugars Starch engineering: DNA sequencing efforts rally occurring polyketides and Cellulosic Bulk NRPs are produced by a number have revealed new metabolic reac5-C sugars biomass chemicals tions and variants of enzymes from of bacteria and fungi using large. (iii) most appropriate microbial host. and detailed models pathway. Their titers and many different organisms. Joint BioEnergy Institute. Unfortunately. E-mail: keasling@berkeley.and processing conditions (e. ways. CA an API is the semisynthesis of the antimalarial 94608. (4. modular enzymes.sciencemag. safe.SPECIALSECTION particularly active pharmaceutical ingredients (APIs). USA. (v) methods cessful for many applications. Berkeley.molecules and hydroxylases to introduce hydroxing the enzymes in the pathway to produce the tion. future production schemes may include designer cells that are tailor-made for the desired all of which are produced through different pathchemical and production process. in every host. different hosts have different terpenes (6–8). these one day be used to produce fine and bulk chemicals.and productivities (Fig. USA. low mostarting materials a large number of chemicals that are currently derived from nonrenewable resources or limited natural resources. readily available. 5). Metabolic engineering has the potential to produce from simple. new genetic tools enable more precise control over metabolic pathengineered industrial hosts (3).resulting enzymes function. In any future. and agrochemicals. metayields in the native producers have chemicals been improved through traditional bolic reactions. More rereactions and design or evolve novel Fuels enzymes for reactions that do not cently. levels of sophistication of genetic tools available. nor will the essential oils. the BIAs are only one of four major alkaloid groups. 2Physical Biosciences Division. duce artemisinin from the microbially sourced 94720. Berkeley. Conversion of sugars to chemicals by means of microbial catalysts. The cost of starting materials is generally a small fraction of their cost. Fig. 5885 Hollis Street. ways.g.. industrial microorganisms. alkaloids are discovered in their natural producers. Unfortunately. 1).2. Since that time. Lawrence Berkeley and Metabolic Routes drug artemisinin with S. 9).3 synthetic pathways for their precursors have been reconstituted in heterologous hosts. Yet even with these substantial de. Department of Chemical and sizable advantage over synthetic organic chem. Isoprenoids are (iv) robust and responsive genetic control system for challenges. as those in synthetic organic chemistry. University of California. and fuels has been enabled by combining enzymes or one day be producible in Escherichia coli and pathways from different hosts into a single microorganism and by engineering enzymes to have new function. Microbial production of natural products has been achieved lecular weight compounds found primarily in and derived from plants and widely used as drugs. Most APIs fall into three classes of natural products. and with continued there are alternative precursor production pathfor debugging and debottlenecking the constructed developments more applications will be possible. microbial catalysts are not as malleable each other: Genes cannot be expressed. Keasling1. taking advantage of terpene synability of starting materials (e. some of which are too complex to be chemically synthesized and yet have a value that justifies the cost of developing a genetically engineered microorganism.org SCIENCE VOL 330 3 DECEMBER 2010 1355 Downloaded from www. CA 94720.but not another host.one of the few classes of natural products where the desired pathways and chosen host.

. including amino acids. and production process all affect host choice. 1. Additionally. there is an opportunity to synthesize multisubstituent APIs (e. sugars from cellulosic biomass). 10–12) and combinatorial expression of these evolved enzymes in a heterologous host will enable the production of unnatural terpenes. and esters) typical of diesel and jet fuel can be produced by way of the fatty acid biosynthetic pathway (17–19). it is now possible to consider production of these inexpensive chemicals from low-cost starting materials such as starch.and six-carbon sugars. 2. This will require simultaneous expression of multiple precursor pathways in a single microorganism. For many of the same reasons that it is desirable to produce petroleum-derived chemicals using biological systems. the fuel producer must be able to consume both five. The benefit would be the synthesis of complicated molecules that might not otherwise be produced. heterologous hosts have been engineered to enhance their production. is now being produced from glucose by E. To make these new fuels economically viable. a useful intermediate in the synthesis of polyurethanes and polyesters. 16). Regulatory proteins or RNAs bind the toxic metabolite and down-regulate the biosynthetic pathway and upregulate the consumption pathway.Metabolism artemisinic acid (6. VOL 330 SCIENCE www. profit margins are generally much lower than for APIs and may be affected by substrate availability and cost. bulk chemicals such as solvents and polymer precursors are rarely produced from microorganisms.. if the host can survive (and thrive) under the desired process conditions (e. For fine chemicals. organic acids. There is an opportunity to produce many other bulk chemicals (e.org 1356 3 DECEMBER 2010 Downloaded from www. Although not as valuable as pharmaceuticals. pH. such as short. starting materials. recent developments in engineering yeast to catabolize these sugars will make production of these fuels more economically viable (22). and if good genetic tools are available to Met Met (-) Gene R Gene A Gene B Gene C (+) Gene 1 Gene 2 Gene 3 Gene P Substrate Metabolite Product Fig. branches in the chain are beneficial— regularly branched and cyclic hydrocarbons of different sizes with diverse structural and chemical properties can be produced via the isoprenoid biosynthetic pathway (20.3-propanediol (1.. etc.g. whereas others have chemical routes. highly branched hydrocarbons (e. flavors. 2011 . more recent work focused on engineering yeasts and bacteria to produce ethanol or butanol from a variety of sugars while eliminating routes to side products and improving the tolerance of the host to the alcohol (14). and a host of other terpene-functionalizing enzymes (8. Although the pathways described above produce a wide range of fuel-like molecules. 9). Yet we have barely begun to investigate what will be possible to produce. branched-chain alcohols can be produced by way of the Ehrlich pathway. it is desirable to produce transportation fuels from readily available. fragrances. etc. For diesel in cold weather and jet fuel at high altitudes. it should be evident that the product. there are many other molecules that one might want to produce.g. 2. ionic strength. some enzymes produce mixtures of products from a single precursor—maybe these enzymes could be tuned to produce a fuel mixture ideal for a particular engine type or climate.. coli engineered with genes from Klebsiella pneumoniae and S. cerevisiae (13).g.4-trimethyl pentane or isooctane) that would be excellent substitutes for petroleumderived gasoline. Some of these molecules are sufficiently complicated that they cannot be produced economically by any route other than biological production. Because many yeasts do not consume fivecarbon sugars. Linear hydrocarbons (alkanes. Beyond producing natural products. vitamins. It should be possible to engineer single microbes or microbial consortia to produce a mixture of fuels from one of the biosynthetic pathways or from multiple biosynthetic pathways. primarily ethanol and butanol. flavors. For some important products (fragrances. terpene hydroxylases. amino acids).sciencemag.). Taxol) or other molecules from the products of multiple biosynthetic pathways. making purification simple and reducing fuel cost. laboratory evolution or rational engineering of terpene cyclases. renewable sources of carbon.g. Larger. Recent advances in metabolic pathway and protein engineering have made it possible to engineer microorganisms to produce hydrocarbons with properties similar or identical to those of petroleum-derived fuels and thus compatible with our existing transportation infrastructure.g. we must tap into inexpensive carbon sources (namely. as well as “ligases” that can assemble multiple substituents together into a single molecule. most petroleum fuels are mixtures of large numbers of components that together create the many important properties of the fuels. and nutraceuticals. Although individual metabolic pathways have been developed to produce natural products derived from a single pathway. alkenes. Hosts and Expression Systems From the applications cited above. In contrast. and political considerations. dedicated energy crops. There is a long history of using microorganisms to produce alcohols. Use of synthetic regulators to modulate metabolic pathways that have a toxic intermediate. For example. agricultural and forest waste. ambient versus extremes of temperature.) with a microbial catalyst.2. sucrose. several microbes have now been engineered to produce these fuels (15. inexpensive. Due to fluctuations in petroleum prices and recognition of dwindling reserves. polymer precursors) by using metabolically engineered cells. but the key will be to produce the exact molecule needed for existing products rather than something “similar but green” that will require extensive product testing before it can be used. Some of the most important qualities one must consider when choosing a host are whether the desired metabolic pathway exists or can be reconstituted in that host.sciencemag. Both the fatty acid– derived and the isoprenoid-derived fuels diffuse (or are pumped) out of the engineered cells and phase separate in the fermentation.. Although much of the work on these alcohols was done by traditional strain mutagenesis and selection.3-PDE). trade imbalances. if the host is genetically stable (both with the introduced pathway and not susceptible to phage attack).org on December 13. By far the highest-volume (and lowestmargin) application for engineered metabolism is the production of transportation fuels. or cellulosic biomass (e. Engineering fuel-producing microorganisms to secrete cellulases and hemicellulases to depolymerize these sugar polymers into sugars before uptake and conversion into fuels has the potential to substantially reduce the cost of producing the fuel. many fine chemicals have been produced economically from natural and engineered microorganisms. because they can be produced inexpensively from petroleum by chemical catalysis. Given the variety of sugars in cellulosic biomass. 7. Indeed. some of which might be more effective than the natural product for the treatment of human disease. By incorporating broad substrate-range 2-keto acid decarboxylases and alcohol dehydrogenases. These alcohols are generally considered better fuels than ethanol and butanol and can also be used to produce a variety of commodity chemicals. 21).

or controlling segmental mRNA stability of each coding region to regulate the amount of each enzyme produced (32). 25). S. Because production of complicated molecules often requires several enzymes. The key issue necessitating good genetic tools is the introduction of foreign genes encoding the metabolic pathway and control over their expression to maximize yields and titers.sciencemag. enzymes. There are many ways to coordinate expression of multiple genes. undefined medium with minimal additions (24. Constitutive promoters (26) and promoters that respond to a change in growth condition or to an important intermediary metabolite (30) allow for inexpensive. The development of yeast IRESs would allow one to express genes encoding metabolic that might otherwise be used to produce the desired molecule of interest. There is growing recognition that one or only a few copies of a gene are needed. and cell CAD software Commercial DNA synthesis FAB Cell production facility . there are many factors other than copy number that can be manipulated to alter enzyme production. Bacillus subtilis. cerevisiae excel in the genetic tools available. The future of engineered biocatalysts. particularly important for production of low-margin chemicals. to name a few. minimal hosts that require addition of many nutrients or that cannot cope with stresses in processing will probably not find a niche in industrial chemical or fuel production where cost is critical. as well as the stabilities of the mRNA (28) and the resulting protein produced from it. Thus. Furthermore. And while B. and the ability to replicate and express large sequences of DNA. while underexpressed genes will create pathway bottlenecks. Widely used. particularly for metabolic engineering applications. One of the limitations to expressing multiple genes in yeast is the lack of internal ribosomal entry sequences (IRESs) that are available for higher eukaryotes. because intermediates of a foreign metabolic pathway can be toxic to the heterologous host (6). etc.sciencemag. but must be produced in catalytic amounts sufficient to adequately transform the metabolic intermediates into the desired products at a sufficient rate. The genes encoding the transformational enzymes in metabolically engineered cells do not need to be highly expressed. subtilis and S. 2011 Enzyme. Promoters play an essential role in controlling biosynthetic pathways. E. coli. which results in decreased production of the desired final compound. coelicolor have the ability to easily express polyketide synthases. With the ability to vary promoter (26) and ribosome binding strength (27). Although there are many inducible promoters for bacteria. it is essential that the relative levels of the enzymes be coordinated. such as using a nonnative RNA polymerase or transcription factor to induce multiple promoters (31).org on December 13. it is desirable to coordinate expression of the genes encoding these enzymes to prevent accumulation of toxic intermediates and bottlenecks in biosynthetic pathways. bacterial hosts may have scientific interest (23). Expression of the desired genes at too high a level will rob the cell of metabolites those elements are synthesized at a FAB and incorporated into a ghost envelope to obtain the new catalyst. cerevisiae or E. Important features of the cloning vector include segregational stability. varying the ribosome binding strength for the enzymes encoded in the operon (27). promoters. 2). related genes into operons. Inducible promoters are one of the easiest and most effective ways to regulate gene expression. Central to any genetic manipulation is the vector used to carry and/or harbor the transforming DNA in the host.org 3 DECEMBER 2010 1357 Downloaded from www. coli. the small number of inducible promoters for yeast and other potential industrial hosts makes regulation of metabolic pathways in those organisms more challenging than in bacteria. and genetic controls are designed from characteristics of parts (enzymes. it is essential to have genetic tools for existing industrial hosts that can grow on simple. inducer-free gene expression. 3.SPECIALSECTION DNA repository and registry Metabolic pathway and cell design Ghost envelope Constructed chromosome Engineered microbial catalyst Fermentation Desire Desired products Fig. but it is essential that the promoter be induced consistently in all cells SCIENCE VOL 330 www. Although E. The design of the engineered catalyst is influenced by the desired product and the production process. coli and S. coli has the disadvantage of being susceptible to phage attack. minimal and consistent copy number in all cells of a culture. Although minimal. they have fewer genetic tools available than either S. cerevisiae. pathway. which is particularly important where cost is an issue (Fig. Pathways. grouping multiple. heterologous hosts include E.) by means of pathway and enzyme CAD software. inexpensive carbon sources and salts or on an inexpensive. and Streptomyces coelicolor. of a culture (29). The chromosomes encoding manipulate the host.

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C.-B. 3.. K. Patent 20070092957 (2007). Gramajo. Opin. manufacturing of molecules will be done with well-known. del Cardayre. 35. G. L. D. J. Lieu. T. Atsumi. 23. B. K. Appl. 27. C. 27. and ease of construction are but a few of the important considerations in designing the chromosome. X. 454 (2003). Y. Designer Cells for Designer Chemicals One can envision a future when a microorganism is tailor-made for production of a specific chemical from a specific starting material. J. Information from one or more of these techniques can be used to diagnose the problem and modify expression of genes in the metabolic pathway or in the host to improve titer and/or productivity. G. J. Biotechnol. J.S. Gottlieb. Biol. 34. H. Lee. Environ.. B. R. Hawkins. E. 274 (2007). 82 (1998). Jensen. G. Nat. 10. Environ. Lee. Analogous to software debuggers that allow one to find and fix errors in computer code. 31. M. M. Eachus. 9. 38. D. 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Science 314. one company builds the membrane and cell wall (the “bag”). S. engineered transporters would be incorporated into the membrane to pump the desired product out of the cell and keep it out and to import the desired starting material. Keasling. B. Safety for the plant operators and the environment would be an essential design criterion. T. In the event that an enzyme does not exist for a particular reaction or set of reactions. Beller. 3.sciencemag. J. Biotechnol. D. Pitera. V. 75. G. Microb. the chromosome would arrive in pieces and be assembled in the constructed envelope or would be completely assembled at the factory and sent to another location to be introduced into the ghost cell. Cane. R. E. D. Acad. In almost all areas of engineering. Admiraal. Ro. 258 (2007). V. K. Withers. 16. 3229 (2008). A. Newman. E. H. D. Nielsen. H.g. 21. Alper. Keasling. Gibson et al. Microbiology 147. C. L. 9. T. 12. Environ. Q. Keasling. 4. M. Farmer. Palsson. D. T. The cell envelope would be designed to be resistant to the specific desired chemical. Ferrin. L. H... Alper. J. because imbalances in a metabolic pathway often elicit a stress response in central metabolism (due to protein overproduction or accumulation of toxic intermediates or end products) (6. Goh. H. Rude.S. Keasling.1193990 1358 3 DECEMBER 2010 VOL 330 SCIENCE www. 23. Keasling. and the cell wall would be designed to make the organism tolerant to industrial processing conditions. C. D. K. Eng. Biol. Proc. S. 106. metabolic engineering should be just as powerful as synthetic chemistry. Microbiol. J. much like chemical engineers build refineries and other chemical factories from unit operations (Fig. D. B. Pronk. K. D. A. Martin. However. Nature 451. ACS Chem. A. 25. D. Wang et al. J. K. 7. 907 (2009). E. 19. Bioeng.. Redundancies in the genetic control system would be engineered to ensure that design parameters are maintained regardless of the transient changes the cell encounters during the production process. Liao. 52 (2010). safe. Debottlenecking. D. there are models and simulation tools that allow one to predict which components to assemble to obtain a larger system with a desired function or characteristic. Science 329. P. including genetic control system failure. Nat. Wanner. Environ. D. Bailey. S. there will always be “bugs” in the engineered system. J. Arnold. Siewers. Sci. operating the engineered cell at the boiling point of volatile. D. 64. Chem. G. Kim. R. van Maris. 18. C. J. Voigt. and together the two disciplines can greatly expand the number of products available from renewable resources. Donaldson. M. J. Renninger. J. Chem. Office of Science. World Patent 200804555 (2008). E. 1565 (2006). H.

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